Immunofluorescence Imaging of GPER and Cytoskeleton

Cells were grown and treated on coverslips, then fixed with a 4% paraformaldehyde Phosphate Buffer Saline solution and incubated with GPER (Santa Cruz, CA, USA; sc-48525), β-tubulin (Cell Signaling, Danvers, MA, USA; 21285S) primary antibodies (1:100 dilution) followed by incubation with Alexa 555 anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA; A21429) (1:1000 dilution) as previously described [38 (link)] or with phalloidin-i-Fluor 488 reagent (Abcam, Cambridge, UK; Ab176753). Finally, the cells were incubated with 1 µg/mL Hoechst 33342 (Sigma-Aldrich). Images were obtained with an ImageXpress (Molecular Devices, San Jose, CA, USA) microconfocal imager.